Co-culture of primary neurons with astrocytes

48 hours before neuron isolation, isolated astrocytes were plated in poly-D-lysine coated, 24 well or 6 well 0.4 μm cell-culture inserts at a density of 50,000 or 1×10⁶ cells/well, respectively. 1 hour after isolated neurons were adhered to plates, astrocyte inserts were replaced with neuronal medium and placed into wells with neuronal cultures. Neuron-astrocyte co-cultures were maintained at 37°C with 5% CO₂ for various times before different treatment, after which plates with neurons and inserts with astrocytes were separated for various analyses.